Stress protein-peptide complexes as prophylactic and therapeutic vaccines against intracellular pathogens

ABSTRACT

Disclosed is a family of vaccines that contain stress protein-peptide complexes which when administered to a mammal are operative to initiate in the mammal a cytotoxic T cell response against cells infected with a preselected intracellular pathogen. Also disclosed are methodologies for preparing and administering vaccines containing such stress protein-peptide complexes.

This is a continuation of application Ser. No. 08/704,727, filed Sep.13, 1996, now U.S. Pat. No. 6,048,530, which is national stage ofPCT/US95/03311, filed Mar. 16, 1995, published in English as WO 95/24923on Sep. 21, 1995, which is a continuation-in-part of application Ser.No. 08/210,421 filed Mar. 16, 1994, now U.S. Pat. No. 5,961,979, each ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of vaccine development.More particularly, the invention relates to the development ofprophylactic and therapeutic vaccines effective against intracellularpathogens.

BACKGROUND OF THE INVENTION

The development of vaccines directed against intracellular pathogens,for example, viruses, bacteria, protozoa, fungi, and intracellularparasites, is ongoing. The development and use of vaccines has provedinvaluable in preventing the spread of disease in man. For example, in1967, smallpox was endemic in 33 countries with 10 to 15 million casesbeing reported annually. At that time, the World Health Organizationintroduced a program to eradicate smallpox. Approximately one decadelater, smallpox was successfully eradicated from the human population.

Theoretically, an ideal vaccine has a long shelf life, is capable ofinducing with a single dose long lasting immunity against a preselectedpathogen and all of its phenotypic variants, is incapable of causing thedisease to which the vaccine is directed against, is effectivetherapeutically and prophylactically, is prepared easily andeconomically using standard methodologies, and can be administeredeasily in the field.

Presently four major classes of vaccine have been developed againstmammalian diseases. These include: live-attenuated vaccines; non livingwhole vaccines; vector vaccines; and subunit vaccines. Several reviewsdiscuss the preparation and utility of these classes of vaccines. Seefor example, Subbarao et al. (1992) in Genetically Engineered Vaccines,edited by Ciardi et al., Plenum Press, New York; and Melnick (1985) inHigh Technology Route to Virus Vaccines, edited by Deesman et al.,published by the American Society for Microbiology, the disclosures ofwhich are incorporated herein by reference. A summary of the advantagesand disadvantages of each of the four classes of vaccines is set forthbelow.

Live attenuated vaccines comprise live but attenuated pathogens, i.e.,non-virulent pathogens, that have been “crippled” by means of geneticmutations. The mutations prevent the pathogens from causing disease inthe recipient or vaccinee. The primary advantage of this type of vaccineis that the attenuated organism stimulates the immune system of therecipient in the same manner as the wild type pathogen by mimicking thenatural infection. Furthermore, the attenuated pathogens replicate inthe vaccinee thereby presenting a continuous supply of antigenicdeterminants to the recipient's immune system. As a result, livevaccines can induce strong, long lasting immune responses against thewild type pathogen. In addition, live vaccines can stimulate theproduction of antibodies which neutralize the pathogen. Also they caninduce resistance to the pathogen at its natural portal of entry intothe host. To date, live attenuated vaccines have been developed against:smallpox; yellow fever; measles; mumps; rubella; poliomyelitis;adenovirus; and tuberculosis.

Live attenuated vaccines, however, have several inherent problems.First, there is always a risk that the attenuated pathogen may revertback to a virulent phenotype. In the event of phenotypic reversion, thevaccine may actually induce the disease it was designed to provideimmunity against. Second, it is expensive and can be impractical todevelop live vaccines directed against pathogens that continuouslychange their antigenic determinants. For example, researchers have beenunable to develop a practical live vaccine against the influenza virusbecause the virus continually changes the antigenic determinants of itscoat proteins. Third, live attenuated vaccines may not be developedagainst infections caused by retroviruses and transforming viruses. Thenucleic acids from these viruses may integrate into the recipientsgenome with the potential risk of inducing cancer in the recipient.Fourth, during the manufacture of live attenuated vaccines adventitiousagents present in the cells in which the vaccine is manufactured may becopurified along with the attenuated pathogen. Alien viruses that havebeen detected in vaccine preparations to date include the avian leukosisvirus, the simian papovavirus SV40, and the simian cytomegalovirus.Fifth, live vaccine preparations can be unstable therefore limitingtheir storage and use in the field. Presently, attempts are being madeto develop stabilizing agents which enhance the longevity of the activevaccines.

Non living whole vaccines comprise non viable whole organisms. Thepathogens are routinely inactivated either by chemical treatment, i.e.,formalin inactivation, or by treatment with lethal doses of radiation.Non living whole vaccines have been developed against: pertussis;typhus; typhoid fever; paratyphoid fever; and particular strains ofinfluenza.

In principle, non living vaccines usually are safe to administer becauseit is unlikely that the organisms will cause disease in the host.Furthermore, since the organism is dead the vaccines tend to be stableand have long shelf lives. There are, however, several disadvantagesassociated with non living whole vaccines. First, considerable care isrequired in their manufacture to ensure that no live pathogens remain inthe vaccine. Second, vaccines of this type generally are ineffective atstimulating cellular responses and tend to be ineffective againstintracellular pathogens. Third, the immunity elicited by non viablevaccines is usually short-lived and must be boosted at a later date.This process repeatedly entails reaching the persons in need ofvaccination and also raises the concern about hypersensitizing thevaccinee against the wild type pathogen.

Vector vaccines, also known as live recombinant vehicle vaccines, may beprepared by incorporating a gene encoding a specific antigenicdeterminant of interest into a living but harmless virus or bacterium.The harmless vector organism is in turn to be injected into the intendedrecipient. In theory, the recombinant vector organism replicates in thehost producing and presenting the antigenic determinant to the host'simmune system. It is contemplated that this type of vaccine will be moreeffective than the non-replicative type of vaccine. For such a vaccineto be successful, the vector must be viable, and be either naturallynon-virulent or have an attenuated phenotype.

Currently preferred vectors include specific strains of: vaccinia(cowpox) virus, adenovirus, adeno-associated virus, salmonella andmycobacteria. Live strains of vaccinia virus and mycobacteria have beenadministered safely to humans in the form of smallpox and tuberculosis(BCG) vaccines, respectively. They have been shown to express foreignproteins and exhibit little or no conversion into virulent phenotypes.Several types of vector vaccines using the BCG vector currently arebeing developed against the human immunodeficiency virus (HIV). Forexample, the HIV antigenic proteins: gag; env; HIV protease; reversetranscriptase; gp120 and gp41 have been introduced, one at a time, intothe BCG vector and shown to induce T cell mediated immune responsesagainst the HIV proteins in animal models (Aldovini et al. (1991) Nature351:479-482; Stover et al. (1991) Nature 351:456-460; Colston (1991)Nature 351:442-443).

Vector vaccines are capable of carrying a plurality of foreign genesthereby permitting simultaneous vaccination against a variety ofpreselected antigenic determinants. For example, researchers haveengineered several HIV genes into the vaccinia virus genome therebycreating multivalent vaccines which therefore are, in theory, capable ofsimultaneously stimulating a response against several HIV proteins.

There are several disadvantages associated with vector vaccines. First,it is necessary to identify suitable strains of viable butnon-pathogenic organisms that may act as carriers for the genes ofinterest. Second, vector vaccines can be prepared only when apotentially protective antigenic determinants has been identified andcharacterized. Accordingly, vector vaccines cannot be prepared againstpathogens whose antigenic determinant has not yet been identified or areso variable that the prospect of identifying the antigenic determinantfor each variant is impractical. Third, the genes encoding thepreselected antigenic determinant must be stably transfected andexpressed in the preferred carrier organism. Consequently, themethodologies required for developing this type of vaccine are bothlabor intensive and time consuming. Fourth, it has not yet beenestablished that recombinant vector vaccines effectively immunize arecipient against a preselected pathogen.

Subunit vaccines usually comprise a subcellular component purified fromthe pathogen of interest. Subunit vaccines usually are safe toadminister because it is unlikely that the subcellular components willcause disease in the recipient. The purified subcellular component maybe either a defined subcellular fraction, purified protein, nucleic acidor polysaccharide having an antigenic determinant-capable of stimulatingan immune response against the pathogen. The antigenic components can bepurified from a preparation of disrupted pathogen. Alternatively, theantigenic proteins, nucleic acids or polysaccharides may be synthesizedusing procedures well known in the art. Diseases that have been treatedwith subunit type vaccines include: cholera; diphtheria; hepatitis typeB; poliomyelitis; tetanus; and specific strains of influenza.

There are, however, several disadvantages associated with subunitvaccines. First, it is important to identify and characterize theprotective antigenic determinant. This can be a labor intensive and timeconsuming process. As a result it may be impractical to develop subunitvaccines against pathogens with highly variable antigenic determinants.Second, subunit vaccines generally are ineffective at stimulatingcytotoxic T cell responses and so they may be ineffective at stimulatingan immune response against intracellular pathogens. Third, the immunityelicited by subunit vaccines is usually short-lived, and like the nonliving whole vaccines must be boosted at a later date therefore raisingthe concern about hypersensitizing the vaccinee against the wild typepathogen.

Heretofore, many of the inactivated whole and subunit vaccines have notbeen sufficiently immunogenic by themselves to induce strong, protectiveresponses. As a result, immunostimulants including, for example,aluminum hydroxide; intact mycobacteria; and/or mycobacterial componentshave been co-administered with these vaccines to enhance the immuneresponse stimulated by the vaccine. Recently, experiments have shownthat mycobacterial heat shock proteins may act as carriers for peptidevaccines thereby enhancing the immunogenidty of the peptides in vivo(Lussow et al. (1991) Eur. J. Immunol. 21:2297-2302). Further studieshave shown that administering a composition to mice comprising anantigenic peptide chemically crosslinked to a purified mycobacterialstress protein stimulates a humoral (antibody mediated) rather than atemporal (cell mediated) response against the antigenic peptide (Barrioset al. (1992) Eur. J. Immunol. 22:1365-1372).

However, because it is generally believed that cellular responses arerequired for immunizing against intracellular pathogens (see forexample, “Advanced Immunology,” Male et al. (1991) Gower MedicalPublishing; Raychaudhuri et al. (1993) Immunology Today 14: 344-348) itis contemplated that conventional subunit and inactivated whole organismvaccines may be ineffective at stimulating immune responses,specifically cytotoxic T cell responses, against intracellularpathogens.

It is an object of the instant invention to provide a safe subunitvaccine comprising a stress protein-peptide complex for administrationto a mammal that is capable of inducing, by means of a cytotoxic T cellresponse, resistance to infection by a preselected intracellularpathogen. The vaccines prepared in accordance with the invention may beused to elicit an immune response against an intracellular pathogenswhose antigenic determinants have been identified, have not yet beenidentified, or where it is impractical to isolate and characterize eachof the antigenic determinants. The vaccines prepared in accordance withthe invention may be prophylactically and therapeutically effectiveagainst preselected pathogens.

Another object of the invention is to provide a method for inducing in amammal resistance to infection by an intracellular pathogen byadministering to the mammal a stress protein-peptide subunit vaccine.Another object is to provide a method for rapidly and cost effectivelyproducing commercially feasible quantities of the stress protein-peptidevaccines from a cell or cell line infected with the intracellularpathogen or alternatively from a cell or cell line transfected with, andexpressing a gene encoding a specific antigenic determinant. Stillanother object is to provide a method for preparing an immunogenicstress protein-peptide subunit vaccine by reconstituting in vitroimmunologically unreactive stress proteins and peptides thereby toproduce immunoreactive complexes capable of stimulating an immuneresponse against a preselected intracellular pathogen.

These and other objects and features of the invention will be apparentfrom the description, drawings, and claims which follow.

SUMMARY OF THE INVENTION

It has now been discovered that a subunit vaccine containing a stressprotein-peptide complex when isolated from cells infected with apreselected intracellular pathogen and then administered to a mammal caneffectively stimulate cellular immune responses against cells infectedwith the same pathogen. Specifically, the immune response is mediatedthrough the cyto toxic T cell cascade which targets and destroys cellscontaining intracellular pathogens.

The vaccines prepared in accordance with the methodologies describedherein provide an alternative approach for stimulating cellular immunitythereby obviating the use of live (attenuated or otherwise)intracellular pathogens. In addition, the vaccines described herein areideal for inducing immune responses against intracellular pathogenshaving either defined or as yet undefined immunogenic determinants.Furthermore the vaccines may be used to induce immune responses againstintracellular pathogens whose antigenic determinants are either diverseor constantly changing thereby making the isolation and characterizationof antigenic determinants impractical.

In a preferred aspect, the invention comprises a vaccine that can beadministered to a mammal for inducing in the mammal a cytotoxic T cellresponse against a preselected intracellular pathogen. Also, it iscontemplated that the vaccines may induce in the mammal, by means of acytotoxic T cell response, resistance to infection by the preselectedintracellular pathogen. The vaccines manufactured in accordance with theprinciples described herein contain an immunogenic stressprotein-peptide complex that is capable of stimulating in the recipienta cytotoxic T cell response directed against cells infected with thepathogen of interest. The complex when combined with a pharmaceuticallyacceptable carrier, adjuvant, or excipient may be administered to amammal using techniques well known in the art.

The term “vaccine”, as used herein, is understood to mean anycomposition containing a stress protein-peptide complex having at leastone antigenic determinant which when administered to a mammal stimulatesin the mammal an immune response against the antigenic determinant.

The term “stress protein” as used herein, is understood to mean anycellular protein which satisfies the following criteria. It is a proteinwhose intracellular concentration increases when a cell is exposed tostressful stimuli, is capable of binding other proteins or peptides, andis capable of releasing the bound proteins or peptides in the presenceof adenosine triphosphate (ATP) or low pH. Stressful stimuli include,but are not limited to, heat shock, nutrient deprivation, metabolicdisruption, oxygen radicals, and infection with intracellular pathogens.

It will be apparent to the artisan upon reading this disclosure thatother recombinant stress proteins, including non native forms, truncatedanalogs, muteins, fusion proteins as well as other proteins capable ofmimicking the peptide binding and immunogenic properties of a stressprotein may be used in the preparation of stress protein-peptidevaccines disclosed herein.

The first stress proteins to be identified were the heat shock proteins(Hsp). As their name suggests, Hsps are induced by a cell in response toheat shock. Three major families of Hsp have been identified and arecalled Hsp60, Hsp70 and Hsp90 because of their respective molecularweights of about 60,70, and 90 kD. Many members of these familiessubsequently were found to be induced in response to other stressfulstimuli, such as those mentioned above.

Stress proteins are found in all prokaryotes and eukaryotes and exhibita remarkable level of evolutionary conservation. For example, DnaK, theHsp70 from E. coli has about 50% amino acid sequence identity with Hsp70proteins from eukaryotes (Bardwell et al. (1984) Proc. Natl. Acad. Sci.81:848-852). The Hsp60 and Hsp90 families also exhibit similarly highlevels of intrafamilial conservation (Hickey et al. (1989) Mol. CellBiol. 9:2615-2626; Jindal (1989) Mol. Cell. Biol. 9:2279-2283). Inaddition, it has been discovered that the Hsp-60, Hsp-70, and Hsp-90families are composed of proteins that are related to the stressproteins in sequence, for example, having greater than 35% amino acididentity, but whose expression levels typically remain unaltered underconditions stressful to the host cell. An example of such a proteinincludes the constitutively expressed cytosolic protein Hsc 70 to whichis related in amino acid sequence to the stress-induced protein Hsp 70.Accordingly, it is contemplated the definition of stress protein, asused herein, embraces other proteins, muteins, analogs, and variantsthereof having at least 35% to 55%, preferably 55% to 75%, and mostpreferably 75% to 95% amino acid identity with members of the threefamilies whose expression levels in a cell are stimulated in response tostressful stimuli.

The term “peptide”, as used herein, is understood to mean any amino acidsequence that is present in a eukaryotic cell infected with anintracellular pathogen but which is not present in a similar cell whenthe cell is not infected with the same pathogen. The definition embracespeptides that not only originate from the pathogen itself but alsopeptides which are synthesized by the infected cell in response toinfection by the intracellular pathogen.

The term “immunogenic stress protein-peptide complex”, as used herein,is understood to mean any complex containing a stress protein and apeptide that is capable of inducing an immune response in a mammal. Thepeptides preferably are non covalently associated with the stressprotein. The complexes may include, but are not limited to,Hsp60-peptide, Hsp70-peptide and Hsp90-peptide complexes. In a preferredaspect of the invention a stress protein belonging to the Hsp90 family,namely gp96 can be used to generate an effective vaccine containing agp96-peptide complex. Since the peptides can be dissociated from thecomplex in the presence of ATP or low pH potentially antigenic peptidescan be isolated from cells infected with a preselected intracellularpathogen. Consequently, the antigenic determinants for potentially anyintracellular pathogen of interest can be identified readily using themethodologies described herein.

The term “cytotoxic T cell”, as used herein, is understood to mean any Tlymphocyte expressing the cell surface glycoprotein marker CD8 that iscapable of targeting and lysing a target cell which bears a class Ihistocompatibility complex on its cell surface and which is infectedwith an intracellular pathogen. The term “cytotoxic T cell response” isunderstood to mean any cytotoxic activity that is mediated by cytotoxicT cells.

As used herein, the term “intracellular pathogen” is understood to meanany viable organism, including, but not limited to, viruses, bacteria,fungi, protozoa and intracellular parasites, capable of existing withina mammalian cell and causing a disease in the mammal.

In a preferred aspect of the invention, the stress protein-peptidevaccines have particular utility in treating human diseases caused byintracellular pathogens. It is contemplated that the vaccines developedusing the principles described herein will be useful in treatingdiseases of other mammals, for example, farm animals including: cattle;horses; goats; sheep; and pigs, and household pets including: cats; anddogs.

Vaccines may be prepared that stimulate cytotoxic T cell responsesagainst cells infected with viruses including, but not limited to,hepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory synctial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsachie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II). Vaccines also may be prepared that stimulate cytotoxicT cell responses against cells infected with intracellular bacteria,including, but not limited to, Mycobacteria, Rickettsia, Mycoplasma,Neisseria and Legionella. Vaccines also may be prepared that stimulatecytotoxic T cell responses against cells infected with intracellularprotozoa, including, but not limited to, Leishmania, Kokzidioa, andTrypanosoma. Vaccines may be prepared that stimulate cytotoxic T cellresponses against cells infected with intracellular parasites including,but not limited to, Chlamydia and Rickettsia.

In another preferred embodiment of the invention, the stress proteinpeptide vaccine may also contain a therapeutically effective amount of acytokine. As used herein, the term “cytokine” is meant to mean anysecreted polypeptide that influences the function of other cellsmediating an immune response. Currently, preferred cytokines include:interleukin-1α(IL-1α), interleukin-1β(IL-1β), interleukin-2 (IL-2),interleukin-3 (IL-3), interleukin4 (IL-4), interleukin-5 (IL-5),interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8),interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11),interleukin-12 (IL-12), interferon a (IFNα), interferon β(IFNβ),interferon γ, (IFNγ), tumor necrosis factor α(TNFα)), tumor necrosisfactor β(TNFβ), granulocyte colony stimulating factor (G-CSF),granulocyte/macrophage colony stimulating factor (GM-CSF), andtransforming growth factor β(TGF-β). It is contemplated that other butas yet undiscovered cytokines may be effective in the invention. Inaddition, conventional antibiotics may be co-administered with thestress protein-peptide complex. The choice of a suitable antibiotic or acombination thereof, however, will be dependent upon the disease inquestion.

It has been discovered that the vaccine stimulates the cytotoxic T cellresponse via the major histocompatibility complex (MHC) class I cascade.Thus, it is contemplated that the cytotoxic T cell response may beenhanced further by co-administering the vaccine with a therapeuticallyeffective amount of one or more of cytokines that potentiate or modulatecytotoxic T cell responses.

Another preferred embodiment, the invention provides a method forstimulating in a mammal a cellular immune response, specifically acytotoxic T cell response, against cells infected with a preselectedintracellular pathogen. The method involves administering to the mammala vaccine made in accordance with the principles disclosed herein in anamount sufficient to elicit in the mammal a cytotoxic T cell responseagainst the preselected intracellular pathogen.

The vaccine may be administered prophylactically to a mammal in order tostimulate in the mammal a cytotoxic T cell response that preventssubsequent infection of the mammal by the intracellular pathogen.Alternatively, the vaccine may be administered therapeutically to amammal having a disease caused by an intracellular pathogen. It iscontemplated that the vaccine may stimulate a cytotoxic T cell responseagainst cells presently infected with the intracellular pathogen.

The dosage and means of administration of the family of stressprotein-peptide vaccines necessarily will depend upon the nature of thecomplex, the intracellular pathogen and the nature of the disease inquestion. The complex should be administered in an amount sufficient toinitiate a cytotoxic T cell response against the intracellular pathogen.In general, the amount of stress protein-peptide complex administeredmay range from about 0.1 to about 1000 micrograms of complex/kg bodyweight of the mammal/immunization, and preferably in the range of about0.5 to 100 micrograms of complex/kg body weight of themammal/immunization. The recipient preferably should be vaccinated fourtimes at weekly intervals. If necessary, the responses may be boosted ata later date by subsequent administration of the vaccine. It iscontemplated, however, that the optimal dosage and vaccination schedulemay be determined empirically for each stress protein-peptide vaccinecomplex by an artisan using conventional techniques well known in theart.

In another aspect, the invention provides a variety of methodologies forpreparing commercially available amounts of the stress-protein peptidevaccines which when administered to a mammal induce in the mammal acytotoxic T cell response against cells infected with a preselectedantigen. In one approach, the stress protein-peptide complex may beharvested using conventional protein purification methodologies from asample of tissue, an isolated cell or immortalized cell line infectedwith the preselected intracellular pathogen, or an isolated cell orimmortalized cell line transfected with, and expressing a gene encodinga preselected antigenic determinant. The purified complex subsequentlymay be stored or combined with a pharmaceutically acceptable carrier foradministration as a vaccine.

Alternatively, the stress protein-peptide complex may be prepared byreconstituting a potentially antigenic peptide and a stress protein invitro. For example, the antigenic peptide may be eluted from either apurified stress protein-peptide complex or a MHC-peptide complex usingmethodologies well known in the art. Specifically, the peptides may beeluted from the stress protein-peptide complex by incubating the complexin the presence of ATP or low pH. Alternatively, the peptides may beeluted from the MHC-peptide complex by incubating the complex in thepresence of trifluoroacetic acid (TFA). The resulting peptides may bepurified by reverse phase HPLC and their amino acid sequences determinedby standard protein sequencing methodologies. Peptides of definedsequence then may be synthesized using conventional peptide synthesismethodologies. Stress proteins may be purified directly from cellsnaturally expressing the stress proteins. Alternatively, recombinantstress proteins, including non native forms, truncated analogs, muteins,fusion proteins as well as other constructs capable of mimicking thepeptide binding and immunogenic properties of stress proteins may beexpressed using conventional recombinant DNA methodologies. For example,a recombinant stress protein may be expressed from recombinant DNA ineither a eukaryotic or prokaryotic expression system and purified fromthe expression system. The two purified components then may be combinedin vitro to generate a synthetic and completely defined stressprotein-peptide complex. The immunogenicity and specificity of therecombinant complexes subsequently may be assayed in vitro and in vivoto identify useful candidate complexes that stimulate cytotoxic T cellresponses against a preselected intracellular pathogen. Once identified,the synthetic complexes may be prepared on any scale, stored as is, orcombined with pharmaceutically acceptable carriers for administration tomammals.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and features of the invention, as wellas the invention itself, may be more fully understood from the followingdescription, when read together with the accompanying drawings, inwhich:

FIG. 1 shows antigen specific cytotoxic T cell activity of splenocytesderived from mice immunized with a gp96-peptide complex harvested fromBALB/c fibroblasts transfected with the nucleoprotein (NP) gene from thePR8 influenza virus. The cytotoxic activity was assayed by the releaseof ⁵¹Cr from BALB/c fibroblasts expressing the NP gene (filled circles),BALB/c fibroblasts expressing the NP gene but treated with the anti-MHCtype I antisera K44 (empty circles) and from the syngeneic non-NP.transfected cell line 5117 (asterisks).

FIG. 2 shows antigen specific cytotoxic T cell activity of splenocytesderived from mice immunized with gp96-peptide complex harvested fromSV40 transformed SVB6 cells. The cytotoxic activity was assayed by therelease of ⁵¹Cr from SVB6 cells (filled circles) and from a non-SV40transformed syngeneic cell line, MCA (empty circles).

FIGS. 3A-3D shows antigen specific cytotoxic T cell activities ofsplenocytes derived from two mice immunized with a reconstitutedHsp70-peptide complex where the peptide has the sequence SLSDLRGYVYQGL(SEQ. ID. NO. 1). Prior to performing the assay, the splenocytes derivedfrom each mouse were stimulated either once (3A and 3C) or twice (3B and3D) in vitro with lethally irradiated cells transfected with, andexpressing the peptide SLSDLRGYVYQGL (SEQ. ID. NO. 1). Cytotoxicactivity was assayed by the release of ⁵¹Cr from EL4 cells expressingthe peptide (filled triangle) and from EL4 cells not expressing thepeptide (empty triangles).

DETAILED DESCRIPTION

The invention is based on the discovery that a stress protein-peptidecomplex when isolated from a eukaryotic cell infected with a preselectedintracellular pathogen and then administered to a mammal can stimulate acytotoxic T cell response directed against cells infected with the samepathogen. This discovery provides a significant advance to the field ofvaccine development.

In accordance with the invention, the aforementioned discovery isexploited to provide a family of vaccines which may be used to immunizemammals against diseases caused by intracellular pathogens. Inprinciple, the vaccines can be prepared against any intracellularpathogen of interest, for example: viruses; bacteria; protozoa; fungi;or intracellular parasites. Generic methodologies useful for preparingvaccines against all of these classes of pathogens are discussed indetail hereinbelow.

As will be appreciated by those skilled in the art, the stressprotein-peptide vaccines described herein have several advantages overthe vaccines currently available. First, the stress protein-peptidevaccines provide an alternative approach for stimulating cellularimmunity and obviate the use of intact intracellular (attenuated orotherwise) pathogens. Second, since the vaccines do not-contain intactorganisms this reduces the risk of causing the disease the vaccine wasdesigned to induce immunity against. Third, the vaccines describedherein are ideal for inducing immune responses against either definedantigenic determinants isolated from an intracellular pathogen or as yetundefined antigenic determinants. Furthermore, vaccines may be preparedthat are effective against pathogens that normally evade the immunesystem by evolving new antigenic coat proteins, i.e., the influenzavirus. Fourth, vaccines of this type may in principle be preparedagainst any intracellular pathogen of interest. Fifth, the vaccines maybe prepared synthetically using the methodologies described hereinafterthereby providing completely defined vaccines that are suitable foradministration to humans.

It is contemplated that the vaccines may be administered eitherprophylactically or therapeutically. When administered prophylacticallythe vaccine may stimulate in the mammal a cytotoxic T cell response thatpermits the vaccinee to resist subsequent infection by the intracellularpathogen. Alternatively, when administered therapeutically the vaccinemay stimulate in the mammal a cytotoxic T cell response against apathogen which is presently infecting and causing disease in the mammal.

The specific component of the vaccine that induces in the recipient aspecific cytotoxic T cell response against the pathogen is a stressprotein-peptide complex. The peptide may be any amino acid sequence thatis present in a eukaryotic cell infected with an intracellular pathogenbut which is not present when such a cell is not infected with the samepathogen. This includes peptides that not only originate from thepathogen itself but also are synthesized by the infected cell inresponse to infection by the intracellular pathogen.

The immunogenic complexes may be purified from any eukaryotic cell,including: whole tissues; isolated cells; and immortalized eukaryoticcell lines infected with the intracellular pathogen. The complexes maybe purified by using conventional protein purification techniques wellknown in the art. For example, it is contemplated that an immunogeniccomplex capable of stimulating a cytotoxic T cell response against theinfluenza virus may be harvested from a eukaryotic cell line that isinfected with the influenza virus.

In addition, it has been found that the peptide can be eluted from thestress protein-complex either in the presence of ATP or low pH. Neitherthe peptide nor the stress protein individually are effective atinducing a cytotoxic T cell response. These experimental conditions,however, may be exploited to isolate peptides from infected cells whichmay contain potentially useful antigenic determinants. Once isolated,the amino acid sequence of each antigenic peptide may be determinedusing conventional amino acid sequencing methodologies. Consequently,the antigenic determinants for potentially any intracellular pathogen ofinterest can be identified readily using the methodologies describedherein. As discussed in detail hereinafter, this property may beexploited in the preparation of completely synthetic vaccines.

Similarly, it has been found that potentially immunogenic peptides maybe eluted from MHC-peptide complexes using techniques well known in theart. See for example, Falk et al. (1990) Nature 348:248-251; Rotzsche etal. (1990) Nature 348:252-254; Elliott et al. (1990) Nature 348:195-197;Falk et al. (1991) Nature 351:290-296, Demotz et al. (1989) Nature334:682-684; Rotzsche et al. (1990) Science 249:283-287, the disclosuresof which are incorporated herein by reference. Although the peptideseluted from the MHC complexes may define a potentially protectiveantigenic determinant, it is appreciated that administration of theisolated peptide in a conventional subunit vaccine may be ineffective atstimulating a cytotoxic T cell response in the recipient. Consequently,it is contemplated that the peptides eluted from MHC-peptide complexesmay be reconstituted with a stress protein, using the methodologiesdescribed herein, thereby to generate a stress protein-peptide complexeffective at stimulating a cytotoxic T cell response capable oftargeting and lysing cells expressing the antigenic peptide.

Stress proteins useful in the practice of the instant invention may bedefined as any cellular protein that satisfies the following criteria.It is a protein whose intracellular concentration increases when a cellis exposed to a stressful stimuli, it is capable of binding otherproteins or peptides, and it is capable of releasing the bound proteinsor peptides in the presence of adenosine triphosphate (ATP) or low pH.

The first stress proteins to be identified were the heat shock proteins(Hsp). As their name implies, Hsps are synthesized by a cell in responseto heat shock To date, three major families of Hsp have been identifiedbased on molecular weight. The families have been called Hsp60, Hsp70and Hsp90 where the numbers reflect the approximate molecular weight ofthe stress proteins in kD. Many members of these families subsequentlywere found to be induced in response to other stressful stimuliincluding, but not limited to, nutrient deprivation, metabolicdisruption, oxygen radicals, and infection with intracellular pathogens.See for example: Welch (May 1993) Scientific American 56-64; Young(1990) Annu. Rev. Immunol. 8:401-420; Craig (1993) Science260:1902-1903; Gething et al. (1992) Nature 355:33-45; and Lindquist etal. (1988) Annu. Rev. Genetics 22:631-677, the disclosures of which areincorporated herein by reference. Accordingly, it is contemplated thatstress proteins belonging to all three families may be useful in thepractice of the instant invention.

The major stress proteins can accumulate to very high levels in stressedcells, but they occur at low to moderate levels in cells that have notbeen stressed. For example, the highly inducible mammalian Hsp70 ishardly detectable at normal temperatures but becomes one of the mostactively synthesized proteins in the cell upon heat shock (Welch et al.(1985), J. Cell. Biol. 101:1198-1211). In contrast, Hsp90 and Hsp60proteins are abundant at normal temperatures in most, but not all,mammalian cells and are further induced by heat (Lai et al. (1984), Mol.Cell. Biol. 4:2802-10; van Bergen en Henegouwen et al. (1987), GenesDev. 1:525-531).

Stress proteins are among the most highly conserved proteins inexistence. For example, DnaK, the Hsp70 from E. coli has about 50% aminoacid sequence identity with Hsp70 proteins from eukaryotes (Bardwell etal. (1984) Proc. Natl. Acad. Sci. 81:848-852). The Hsp60 and Hsp90families also show similarly high levels of intrafamilial conservation(Hickey et al. (1989) Mol. Cell Biol. 9:2615-2626; Jindal (1989) Mol.Cell. Biol. 9:2279-2283). In addition, it has been discovered that theHsp60, Hsp70 and Hsp90 families are composed of proteins that arerelated to the stress proteins in sequence, for example, having greaterthan 35% amino acid identity, but whose expression levels typicallyremain unaltered under conditions stressful to the host cell. An exampleof such a protein includes the constitutively expressed cystolic proteinHsc 70 which is related in amino acid sequence to the stress-inducedprotein Hsp 70. It is, therefore, contemplated that the definition ofstress protein, as used herein, embraces other proteins, muteins,analogs, and variants thereof having at least 35% to 55%, preferably 55%to 75%, and most preferably 75% to 95% amino acid identity with membersof the three families whose expression levels in a cell are enhanced inresponse to a stressful stimulus. The purification of stress proteinsbelonging to these three families is described below.

The immunogenic stress protein-peptide complexes of the invention mayinclude any complex containing a stress protein and a peptide that iscapable of inducing an immune response in a mammal. The peptidespreferably are non covalently associated with the stress protein.Preferred complexes may include, but are not limited to, Hsp60-peptide,Hsp70-peptide and Hsp90-peptide complexes. For example, a stress proteincalled gp96 which is present in the endoplasmic reticulum of eukaryoticcells and is related to the cytoplasmic Hsp90s can be used to generatean effective vaccine containing a gp96-peptide complex.

Another family of low molecular weight heat shock proteins has now beenidentified and is called Hsp 25/Hsp 27. The purification of theseproteins is discussed below. It is contemplated that these low molecularweight proteins may also have utility in the instant invention.

It has been discovered also that the stress protein-peptide complexes ofthe invention can be prepared from cells infected with an intracellularpathogen as well as cells that have been transformed by an intracellularpathogen. For example, immunogenic stress protein peptide complexes maybe isolated from eukaryotic cells transformed with a transforming virussuch as SV40, see below.

In a preferred aspect of the invention, the purified stressprotein-peptide vaccines may have particular utility in the treatment ofhuman diseases caused by intracellular pathogens. It is appreciated,however, that the vaccines developed using the principles describedherein will be useful in treating diseases of other mammals, forexample, farm animals including: cattle; horses; sheep; goats; and pigs,and household pets including: cats; and dogs, that similarly are causedby intracellular pathogens.

In accordance with the methods described herein, vaccines may beprepared that stimulate cytotoxic T cell responses against cellsinfected with viruses including, but not limited to, hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,HSV-I, HSV-II, rinderpest rhinovirous, echovirus, rotavirus, respiratorysynctial virus, papilloma virus, papova virus, cytomegalovirus,echinovirus, arbovirus, huntavirus, coxsachie virus, mumps virus,measles virus, rubella virus, polio virus, HIV-I, and HIV-II. Similarly,vaccines may also be prepared that stimulate cytotoxic T cell responsesagainst cells infected with intracellular bacteria, including, but notlimited to, Mycobacteria, Rickettsia, Mycoplasma, Neisseria andLegionella. In addition, vaccines may also be prepared that stimulatecytotoxic T cell responses against cells infected with intracellularprotozoa, including, but not limited to, Leishmania, Kokzidioa, andTrypanosoma. Furthermore, vaccines may be prepared that stimulatecytotoxic T cell responses against cells infected with intracellularparasites including, but not limited to, Chlamydia and Rickettsia.

I. Propagation of Infected Eukaryotic Cells.

As will be appreciated by those skilled in the art, the protocolsdescribed herein may be used to isolate stress protein-peptide complexesfrom any eukaryotic cell, for example, tissues, isolated cells orimmortalized eukaryotic cell lines infected with a preselectedintracellular pathogen.

When immortalized animal cell lines are used as a source of the stressprotein-peptide complex it is of course important to use cell lines thatcan be infected with the pathogen of interest. In addition, it ispreferable to use cells that are derived from the same species as theintended recipient of the vaccine.

For example, in order to prepare a stress protein-peptide complex foradministration to humans that may be effective against HIV-I, the virusmay be propagated in human cells which include, but are not limited to,human CD4+ T cells, HepG2 cells, and U937 promonocytic cells. In orderto prepare a stress protein-peptide complex for administration to humansthat may be effective against HIV-II, the virus may be propagated in,for example, human CD4+ T cells. Similarly, influenza viruses may bepropagated in, for example, human fibroblast cell lines and MDCK cells,and mycobacteria may be cultured in, for example, human Schwaan cells.

If the intracellular pathogens do not lyse the infected cells then theinfected cells are cultured under the same conditions as the normaluninfected cells. For example, mycobacteria may be propagated in nervecultures of the sensory ganglia of newborn Swiss white mice. The nervecells are cultured in a growth medium containing 70% Dulbecco modifiedEagle minimal essential medium (DMEM) with 0.006% glucose, 20% fetalcalf serum, 10% chicken embryo extract and cytosine arabinoside. Aftereight to ten days, the cultures are inoculated with 5-8×10⁶ mycobacteriaisolated from fresh nodules of untreated lepromatous leprosy patients.The infected cells may be cultured at 370° C., for up to 6 weeks, afterwhich the infected cells are harvested and the stress protein-peptidecomplexes isolated. See for example, Mukherjee et al. (1985) J. Clin.Micro. 21:808-814, the disclosure of which is incorporated herein byreference.

If, on the other hand, the host cells are lysed by the pathogen ofinterest (as in the case of influenza virus) the cells may still begrown under standard conditions except the cells are washed andharvested just prior to lysis of the host cell. For example, during thepurification of stress protein-peptide complexes from influenza infectedcells, fibroblasts (or other cell types) are infected for 1 hour at 37°C. with 5-10 plaque forming units (PFU) of virus per cell. The infectedcells may be cultured in plain DMEM medium for 24 hours at 37° C. After24 hours the cells are washed and harvested prior to lysis. The stressprotein-peptide complexes may be isolated using the procedures set forthbelow.

In addition, when the gene encoding a particular antigenic determinanthas been identified, the gene of interest may be transfected andexpressed in an immortalized human or other mammalian cell line usingtechniques well known in the art. See for example “Current Protocols inMolecular Biology” (1989), eds. Ausubel F M, Brent R, Kingston R E,Moore D D, Seidman J G, Smith J A and Struhl K, Wiley Interscience, thedisclosure of which is incorporated by reference herein. The transfectedcells may be grown under standard conditions and the complexes isolatedsubsequently.

II. Preparation of Stress Proteins and Immunogenic StressProtein-peptide Complexes

Methods for preparing Hsp70-peptide complexes, Hsp90-peptide complexes,gp96-peptide complexes, Hsp70, Hsp25/Hsp27, and Hsp60 are set forthbelow.

(a) Purification of Hsp70-peptide Complexes.

A pellet of infected cells is resuspended in 3 volumes of 1× Lysisbuffer consisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2mM CaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF).The pellet is sonicated, on ice, until >99% cells are lysed as judged bymicroscopic examination. Alternatively, the cells may be lysed bymechanical shearing. In this procedure, the cells are resuspended in 30mM sodium bicarbonate pH 7.5, 1 mM PMSF, incubated on ice for 20 min.and then homogenized in a dounce homogenizer until >95% cells are lysed.

The lysate is centrifuged at 1000 g for 10 minutes to remove unbrokencells, nuclei and other debris. The supernatant from this centrifugationstep is then recentrifuged at 100,000 g for 90 minutes.

The supernatant is mixed for 2-3 hours at 4° C. with Con A Sepharoseequilibrated with PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cellsare lysed by mechanical shearing, the supernatant is diluted with equalvolume of 2× Lysis Buffer before proceeding. Then the slurry is packedinto a column and washed with 1× lysis buffer. The material that doesnot bind is dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.The dialyzate is centrifuged for 20 min. at 17,000 rpm (Sorvall SS34rotor) and the resulting supernatant applied to a Mono Q FPLC column(Pharmacia) equilibrated in 20 mM Tris-Acetate pH 7.5, 20 mM NaCl, 0.1mM EDTA and 15 mM 2-mercaptoethanol. Then the proteins are eluted with a20 mM to 500 mM NaCl gradient. The fractions are characterized by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andimmunoblotting using an appropriate anti-Hsp70 antibody (such as cloneN27F3-4 from StressCen).

The fractions that are strongly immunoreactive with the antibody arepooled and the Hsp70-peptide complexes precipitated with ammoniumsulfate. The complex is precipitated in the 50%-70% ammonium sulfatecut. The protein pellet is harvested by centrifugation at 17,000 rpm(SS34 Sorvall rotor) and washed with 70% ammonium sulfate. Then thepellet is solubilized and the residual ammonium sulfate removed by gelfiltration on a Sephadex® G25 column (Pharmacia).

The Hsp70-peptide complex can be purified to apparent homogeneity usingthis method. Up to 1 mg of Hsp70-peptide complex can be purified from 1g of cells/tissue.

(b) Purification of Hsp70.

The Hsp70 polypeptide may be purified from the Hsp70-peptide complex byATP agarose chromatography. See for example, Welch et al. (1985) Mol.Cell. Biol. 5:1229, the disclosure of which is incorporated herein byreference. Briefly, MgC₂ is added to the previously isolated complex toa final concentration of 3 mM. Then, the complex is applied to an ATPagarose column (Sigma Chemical Co.) equilibrated in 20 mM Tris-Acetate(pH 7.5), 20 mM NaCl, 0.1 mM EDTA, 15 mM 2-mercaptoethanol, 3 mM MgCl₂.The column is washed extensively with the equilibration buffercontaining 0.5M NaCl, and then washed with buffer without the NaCl. Thenthe Hsp70 is eluted from the column with equilibration buffer containing3 mM ATP (Sigma Chemical Co.).

(c) Purification of Hsp90-peptide complexes.

A pellet of infected cells is resuspended in 3 volumes of 1× Lysisbuffer consisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2mM CaCl2, 2 mM MgCl₂ and 1 mM PMSF. The cell pellet is sonicated, onice, until >95% cells are lysed as judged by microscopic examination.Alternatively, the cells may be lysed by mechanical shearing, as before.

The lysate is centrifuged at 1000 g for 10 minutes to remove unbrokencells, nuclei and other debris. The supernatant from this centrifugationstep subsequently is recentrifuged at 100,000 g for 90 minutes.

Then, the supernatant is mixed for 2-3 hours at 4° C. with Con ASepharose equilibrated with PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. Whenthe cells are lysed by mechanical shearing, the supernatant is dilutedwith equal volume of 2× Lysis Buffer before proceeding. Then, the slurryis packed into a column and washed with 1× lysis buffer. The materialthat does not bind is dialyzed for 36 hours (three times, 100 volumeseach time) against 20 mM sodium phosphate pH 7.4, 1 mM EDTA, 250 mMNaCl, 1 mM PMSF. The dialyzate is centrifuged at 17,000 rpm (SorvallSS34 rotor) for 20 min. The resulting supernatant is applied to a Mono QFPLC column (Pharmacia) equilibrated with lysis buffer and the boundproteins eluted with a salt gradient of 200 mM to 600 mM NaCl.

The eluted fractions are analyzed by SDS-PAGE and the Hsp90 complexesidentified by immunoblotting using an anti-Hsp90 antibody (for example,3G3 from Affinity Bioreagents). Hsp90 can be purified to apparenthomogeneity using this procedure. Approximately 150-200 μg of Hsp90 canbe purified routinely from 1 g of cells/tissue.

(d) Purification of gp96-peptide complexes.

A pellet of infected cells is resuspended in 4 volumes of bufferconsisting of 30 mM sodium bicarbonate buffer (pH7.5) and 1 mM PMSF andthe cells allowed to swell on ice for 20 min. The cell pellet then ishomogenized in a Dounce homogenizer (the appropriate clearance of thehomogenizer will vary according to each cells type) on ice until >95%cells are lysed.

The lysate is centrifuged at 1000 g for 10 minutes to remove unbrokencells, nuclei and other debris. The supernatant from this centrifugationstep then is recentrifuged at 100,000 g for 90 minutes. The gp96-peptidecomplex can be purified either from the 100,000 g pellet or from thesupernatant.

When purified from the supernatant, the supernatant is diluted withequal volume of 2× Lysis Buffer and the supernatant mixed for 2-3 hoursat 4° C. with Con A Sepharose equilibrated with PBS containing 2 mM Ca²⁺and 2 mM Mg²⁺. Then, the slurry is packed into a column and washed with1× lysis buffer until the OD₂₈₀ drops to baseline. Then, the column iswashed with ½ column bed volume of 10% α-methyl mannoside (α-MM)dissolved in PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺, the column sealedwith a piece of parafilm, and incubated at 37° C. for 15 min. Then thecolumn is cooled to room temperature and the parafilm removed from thebottom of the column. Five column volumes of the α-MM buffer are appliedto the column and the eluate analyzed by SDS-PAGE. Typically theresulting material is about 60-95% pure, however this depends upon thecell type and the tissue-to-lysis buffer ratio used. Then the sample isapplied to a Mono Q FPLC column (Pharmacia) equilibrated with a buffercontaining 5 mM sodium phosphate, pH7. The proteins then are eluted fromthe column with a 0-1M NaCl gradient and the gp96 fraction elutesbetween 400 mM and 550 mM NaCl.

This procedure, however, may be modified by two additional steps, usedeither alone or in combination, to consistently produce apparentlyhomogeneous gp96-peptide complexes. One optional step involves anammonium sulfate precipatation prior to the Con A purification step andthe other optional step involves DEAE-Sepharose purification after theCon A purification step but before the Mono Q FPLC step.

In the first optional step, the supernatant resulting from the 100,000 gcentrifugation step is brought to a final concentration of 50% ammoniumsulfate by the addition ammonium sulfate. The ammonium sulfate is addedslowly while gently stirring the solution in a beaker placed in a trayof ice water. The solution is stirred for about 2 to 12 h. at 4° C. andthe resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor).The supernatant resulting from this step is removed, brought to 70%ammonium sulfate saturation by the addition of ammonium sulfatesolution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor). Theresulting pellet from this step is harvested and suspended in PBScontaining 70% ammonium sulfate in order to rinse the pellet. Thismixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pelletdissolved in PBS containing 2 mM Ca² and Mg². Undissolved material isremoved by a brief centrifugation at 15,000 rpm (Sorvall SS34 rotor).Then, the solution is mixed with Con A Sepharose and the procedurefollowed as before.

In the second optional step, the gp96 containing fractions eluted fromthe Con A column are pooled and the buffer exchanged for 5 mM sodiumphosphate buffer, pH 7,300 mM NaCl by dialysis, or preferably by bufferexchange on a Sephadex G25 column. After buffer exchange, the solutionis mixed with DEAE-Sepharose previously equilibrated with 5 mM sodiumphosphate buffer, pH 7,300 mM NaCl. The protein solution and the beadsare mixed gently for 1 hour and poured into a column. Then, the columnis washed with 5 mM sodium phosphate buffer, pH 7,300 mM NaCl, until theabsorbance at 280 nM drops to baseline. Then, the bound protein iseluted from the column with five volumes of 5 mM sodium phosphatebuffer, pH 7, 700 mM NaCl. Protein containing fractions are pooled anddiluted with 5 mM sodium phosphate buffer, pH 7 in order to lower thesalt concentration to 175 mM. The resulting material then is applied tothe Mono Q FPLC column (Pharmacia) equilibrated with 5 mM sodiumphosphate buffer, pH 7 and the protein that binds to the Mono Q FPLCcolumn (Pharmacia) is eluted as described before.

It is appreciated, however, that one skilled in the art may assess, byroutine experimentation, the benefit of incorporating the optional stepsinto the purification protocol. In addition, it is appreciated also thatthe benefit of adding each of the optional steps will depend upon thesource of the starting material.

When the gp96 fraction is isolated from the 100,000 g pellet, the pelletis suspended in 5 volumes of PBS containing either 1% sodiumdeoxycholate or 1% octyl glucopyranoside (but without the Mg²⁺ and Ca²⁺)and incubated on ice for 1 h. The suspension is centrifuged at 20,000 gfor 30 min and the resulting supernatant dialyzed against severalchanges of PBS (also without the Mg²⁺ and Ca²⁺) to remove the detergent.The dialysate is centrifuged at 100,000 g for 90 min, the supernatantharvested, and calcium and magnesium are added to the supernatant togive final concentrations of 2 mM, respectively. Then the sample ispurified by either the unmodified or the modified method for isolatinggp96-peptide complex from the 100,000 g supernatant, see above.

The gp96-peptide complexes can be purified to apparent homogeneity usingthis procedure. About 10-20 μg of gp96 can be isolated from 1 gcells/tissue.

(e) Purification of HSP25 and HSP27.

The purification of Hsp25 and Hsp27 polypeptides has been disclosedpreviously and so is not discussed in detail herein. See for exampleJakob et al. (1993) J. Biol. Chem. 268:1517-1520, the disclosure ofwhich is incorporated herein by reference.

Briefly, the cell lysates are precipitated with 35% ammonium sulfate.The pellet is harvested by centrifugation, solubilized in buffer andfractionated by ion exchange chromatography using a DEAE Sepharose CL-6Bcolumn (Pharmacia Biotechnology, Inc.). The proteins are eluted with50-200 mM NaCl gradient. The fractions containing Hsp25 and Hsp27 areidentified by immunoblotting using suitable antibodies. The fractionsare combined and fractionated by size exclusion chromatography on aSuperose 6 gel filtration column (Pharmacia).

(f) Purification of Hsp60.

The purification of Hsp60 has been discussed in detail previously and sois not discussed in detail herein. See for example, Vitanen et al.(1992) J. Biol. Chem. 267: 695-698, the disclosure of which isincorporated herein by reference.

Briefly, a mitochondrial matrix lysate is applied to a Mono Q FPLCcolumn equilibrated with 50 mM sodium phosphate, 1 mM MgCl₂, 1 mM EGTA,pH 6.9. The proteins are eluted with a 0-1-M NaCl gradient. Thefractions containing Hsp65 are pooled and fractionated by ATP agarosechromatography as discussed above.

III. Preparation of Recombinant Stress Proteins

It is contemplated that recombinant stress proteins and amino acidsequence variants thereof may be prepared using conventional recombinantDNA methodologies. For example, recombinant DNAs encoding either a knownstress protein or a homologue can be inserted into a suitable host cell,the protein expressed, harvested, renatured if necessary, and purified.Stress proteins currently known in the art are summarized in Table I,below.

The processes for manipulating, amplifying, and recombining DNA whichencode amino acid sequences of interest are generally well known in theart, and therefore, not described in detail herein. Methods ofidentifying and isolating genes encoding members of the stress proteinfamilies also are well understood, and are described in the patent andother literature.

Accordingly, the construction of DNAs encoding biosynthetic constructsas disclosed herein can be performed using known techniques involvingthe use of various restriction enzymes which make sequence specific cutsin DNA to produce blunt ends or cohesive ends, DNA ligases, techniquesenabling enzymatic addition of sticky ends to blunt-ended DNA,construction of synthetic DNAs by assembly of short or medium lengtholigonucleotides, cDNA synthesis techniques, and synthetic probes forisolating genes of members of the stress protein families. Variouspromoter sequences and other regulatory DNA sequences used in achievingexpression, and various types of host cells are also known andavailable. Conventional transfection techniques, and equallyconventional techniques for cloning and subcloning DNA are useful in thepractice of this invention and known

TABLE 1 Families of Stress Proteins from Gething, et al., InfraOrganism/Organelle Hsp 60 Hsp 70 Hsp 90 E. coli GroEL DnaK HtpG (C62.5)Yeast /cytosol Ssal-4p Hsp 83/Hsc83 /endoplasmic reticulum Karp2 (BiP)/mitochondria Hsp 60 Ssclp (Mif4p) Drosophila Hsp 68 Hsp 70 Hsc 1.2.4Mammals /cytosol Hsp 70 (p73) Hsp 90 (Hsp83) Hsc 70 (p72) Hsp 87/endoplasmic reticulum BiP (Grp 78) Grp 94 (Erp99) gp96 /mitochondriaHsp 60 Hsp 70 (Grp 75) (Hsp 8) Plants /endoplasmic reticulum b70 (BiP)/chloroplasts RUSBP Alternative names are shown in parentheses.

to those skilled in the art. Various types of vectors may be used suchas plasmids and viruses including animal viruses and bacteriophages. Thevectors may exploit various marker genes which impart to a successfullytransfected cell a detectable phenotypic property that-can be used toidentify which of a family of clones has successfully incorporated therecombinant DNA of the vector.

DNA molecules encoding potentially useful stress proteins may beobtained by a variety of methods. Genes of interest may be purified fromstandard cDNA libraries using colony or plaque hybridizationtechnologies or by using polymerase chain reaction (PCR) methodologies,all of which are well known in the art. See for example, “MolecularCloning: A Laboratory Manual, 2nd Edition” Sambrook et al. (1989), ColdSpring Harbor Press, the disclosure of which is incorporated herein byreference. Alternatively, the preferred genes can be generated by theassembly of synthetic oligonucleotides produced in a conventional,automated, polynucleotide synthesizer followed by ligation withappropriate ligases. For example, overlapping, complementary DNAfragments comprising 15 bases may be synthesized semi manually usingphosphoramidite chemistry, with end segments left unphosphorylated toprevent polymerization during ligation. One end of the synthetic DNA isleft with a “sticky end” corresponding to the site of action of aparticular restriction endonuclease, and the other end is left with anend corresponding to the site of action of another restrictionendonuclease. Alternatively, this approach can be fully automated. TheDNA encoding the biosynthetic constructs may be created by synthesizinglonger single strand fragments (e.g., 50-100 nucleotides long) in, forexample, an Applied Biosystems oligonucleotide synthesizer, and thenligating the fragments.

The recombinant DNA constructs then may be integrated into an expressionvector and transfected into an appropriate host cell for proteinexpression. Useful host cells include E. coli, Saccharomyces theinsect/baculovirus cell system, myeloma cells, and various othermammalian cells. In E. coli and other microbial hosts, the syntheticgenes can be expressed as fusion proteins. Expression in eukaryotes canbe accomplished by the transfection of DNA sequences encoding thebiosynthetic protein of interest into myeloma or other type of cellline.

The vector additionally may include various sequences to promote correctexpression of the recombinant protein, including transcriptionalpromoter and termination sequences, enhancer sequences, preferredribosome binding site sequences, preferred mRNA leader sequences,preferred protein processing sequences, preferred signal sequences forprotein secretion, and the like. The DNA sequence encoding the gene ofinterest also may be manipulated to remove potentially inhibitingsequences or to minimize unwanted secondary structure formation. Therecombinant protein also may be expressed as a fusion protein. Afterbeing translated, the protein may be purified from the cells themselvesor recovered from the culture medium.

For example, if the gene is to be expressed in E. coli, it may first becloned into an expression vector. This is accomplished by positioningthe engineered gene downstream of a promoter sequence such as Trp orTac, and a gene coding for a leader peptide such as fragment B ofprotein A (FB). The resulting fusion proteins accumulate in refractilebodies in the cytoplasm of the cells, and may be harvested afterdisruption of the cells by French press or sonication. The refractilebodies are solubilized, and the expressed proteins refolded and cleavedby methods already established for many other recombinant proteins.

Expression of the engineered genes in eukaryotic cells requires theestablishment of appropriate cells and cell lines that are easy totransfect, are capable of stably maintaining foreign DNA with anunrearranged sequence, and which have the necessary cellular componentsfor efficient transcription, translation, post-translation modification,and secretion of the protein. In addition, a suitable vector carryingthe gene of interest also is necessary. DNA vector design fortransfection into mammalian cells should include appropriate sequencesto promote expression of the gene of interest as described supra,including appropriate transcription initiation, termination, andenhancer sequences, as well as sequences that enhance translationefficiency, such as the Kozak consensus sequence. Preferred DNA vectorsalso include a marker gene and means for amplifying the copy number ofthe gene of interest. A detailed review of the state of the art of theproduction of foreign proteins in mammalian cells, including usefulcells, protein expression-promoting sequences, marker genes, and geneamplification methods, is disclosed in Genetic Engineering 7:91-127(1988).

The best-characterized transcription promoters useful for expressing aforeign gene in a particular mammalian cell are the SV40 early promoter,the adenovirus promoter (AdMLP), the mouse metallothionein-I promoter(mMT-I), the Rous sarcoma virus (RSV) long terminal repeat (LTR), themouse mammary tumor virus long terminal repeat (MMTV-LTR), and the humancytomegalovirus major intermediate-early promoter (hCMV). The DNAsequences for all of these promoters are known in the art and areavailable commercially.

The use of a selectable DHFR gene in a dhfr cell line is a wellcharacterized method useful in the amplification of genes in mammaliancell systems. Briefly, the DHFR gene is provided on the vector carryingthe gene of interest, and addition of increasing concentrations of thecytotoxic drug methotrexate leads to amplification of the DHFR gene copynumber, as well as that of the associated gene of interest. DHFR as aselectable, amplifiable marker gene in transfected Chinese hamster ovarycell lines (CHO cells) is particularly well characterized in the art.Other useful amplifiable marker genes include the adenosine deaminase(ADA) and glutamine synthetase (GS) genes.

The choice of cells/cell lines is also important and depends on theneeds of the experimenter. Monkey kidney cells (COS) provide high levelsof transient gene expression, providing a useful means for rapidlytesting vector construction and the expression of cloned genes. COScells are transfected with a simian virus 40 (SV40) vector carrying thegene of interest. The transfected COS cells eventually die, thuspreventing the long term production of the desired protein product.However, transient expression does not require the time consumingprocess required for the development of a stable cell line. Amongestablished cell lines, CHO cells may be the best-characterized to date.CHO cells are capable of expressing proteins from a broad range of celltypes. The general applicability of CHO cells and its successfulproduction for a wide variety of human proteins in unrelated cell typesemphasizes the underlying similarity of all mammalian cells.

The various cells, cell lines and DNA sequences that can be used formammalian cell expression of the recombinant stress protein constructsof the invention are well characterized in the art and are readilyavailable. Other promoters, selectable markers, gene amplificationmethods and cells also may be used to express the proteins of thisinvention. Particular details of the transfection, expression, andpurification of recombinant proteins are well documented in the art andare understood by those having ordinary skill in the art. Furtherdetails on the various technical aspects of each of the steps used inrecombinant production of foreign genes in mammalian cell expressionsystems can be found in a number of texts and laboratory manuals in theart, such as, for example, Current Protocols in Molecular Biology,(1989) eds. Ausubel et al., Wiley Interscience.

IV. Isolation of Potentially Immunogenic Peptides

As mentioned previously, potentially immunogenic peptides may beisolated from either stress protein-peptide complexes or MHC-peptidecomplexes. Protocols for isolating peptides from either of thesecomplexes are set forth below.

(a) Peptides from Stress Protein-peptide Complexes.

Two methods may be used to elute the peptide from a stressprotein-peptide complex. One approach involves incubating the stressprotein-peptide complex in the presence of ATP, the other involvesincubating the complexes in a low pH buffer.

Briefly, the complex of interest is centrifuged through a Centricon 10assembly (Millipore) to remove any low molecular weight material looselyassociated with the complex. The large molecular weight fraction may beremoved and analyzed by SDS-PAGE while the low molecular weight may beanalyzed by HPLC as described below. In the ATP incubation protocol, thestress protein-peptide complex in the large molecular weight fraction isincubated with 10mM ATP for 30 minutes at room temperature. In the lowpH protocol, acetic acid is added to the stress protein-peptide complexto give a final concentration of 10% (vol/vol) and the mixture incubatedin a boiling water bath for 10 minutes. See for example, Van Bleek etal. (1990) Nature 348:213-216; and Li et al. (1993) EMBO Journal12:3143-3151, the disclosures of which are incorporated herein byreference.

The resulting samples are centrifuged through an Centricon 10 assemblyas mentioned previously. The high and low molecular weight fractions arerecovered. The remaining large molecular weight stress protein-peptidecomplexes can be reincubated with ATP or low pH to remove any remainingpeptides.

The resulting lower molecular weight fractions are pooled, concentratedby evaporation and dissolved in 0.1% trifluoroacetic acid (TFA). Then,the dissolved material is fractionated by reverse phase high pressureliquid chromatography (HPLC), using for example a VYDAC C18 reversephase column equilibrated with 0.1% TFA. The bound material subsequentlyis eluted by developing the column with a linear gradient of 0 to 80%acetonitrile in 0.1% TFA at a flow rate of about 0.8 ml/min. The elutionof the peptides can be monitored by OD₂₁₀ and the fractions containingthe peptides collected.

(b) Peptides from MHC-peptide Complexes.

The isolation of potentially immunogenic peptides from MHC molecules iswell known in the art and so is not described in detail herein. See forexample, Falk et al. (1990) Nature 348:248-251; Rotzsche et al. (1990)Nature 348:252-254; Elliott et al. (1990) Nature 348:195-197; Falket al.(1991) Nature 351:290-296, Demotz et al. (1989) Nature 343:682-684;Rotzsche et al. (1990) Science 249:283-287.

Briefly, MHC-peptide complexes may be isolated by a conventionalimmunoaffinity procedure. Then the peptides are eluted from theMHC-peptide complex by incubating the complexes in the presence of about0.1% TFA in acetonitrile. The extracted peptides may be fractionated andpurified by reverse phase HPLC, as before.

The amino acid sequences of the eluted peptides may be determined eitherby manual or automated amino acid sequencing techniques well known inthe art. Once the amino acid sequence of a potentially protectivepeptide has been determined the peptide may be synthesized in anydesired amount using conventional peptide synthesis or other protocolswell known in the art.

V. Synthesis of Potentially Useful Immunogenic Pep tides

Peptides having the same amino acid sequence as those isolated above maybe synthesized by solid-phase peptide synthesis using procedures similarto those described by Merrifield (1963) J. Am. Chem. Soc., 85:2149.During synthesis, N-α-protected amino acids having protected side chainsare added stepwise to a growing polypeptide chain linked by itsC-terminal end to an insoluble polymeric support i.e., polystyrenebeads. The peptides are synthesized by linking an amino group of anN-α-deprotected amino acid to an α-carboxy group of an N-α-protectedamino acid that has been activated by reacting it with a reagent such asdicyclohexylcarbodiimide. The attachment of a free amino group to theactivated carboxyl leads to peptide bond formation. The most commonlyused N-α-protecting groups include Boc which is acid labile and Fmocwhich is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe polystyrene beads. The N-α-protecting group is then removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-α-protected amino acid. The process is repeateduntil the desired peptide is synthesized. The resulting peptides thenare cleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides can be derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino adds and reagents are well known inthe art and so are not discussed in detail herein. See for example,Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach,IRL Press, (1989), and Bodanszky, Peptide Chemistry, A PracticalTextbook, 2nd Ed, Springer-Berlog (1993), the disclosures of which areincorporated herein by reference.

Purification of the resulting peptides is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

VI. Reconstitution of Stress Protein-peptide Complexes

As will be appreciated by those skilled in the art, the peptides, eitherisolated from the complexes using the aforementioned procedures orchemically synthesized, may be reconstituted with a variety of naturallypurified or recombinant stress proteins in vitro to generate immunogenicstress protein-peptide complexes. A preferred protocol forreconstituting a stress protein and a peptide in vitro is discussedbelow.

Prior to reconstitution the stress proteins are pretreated with ATP orlow pH to remove any peptides that may be associated with thestress-protein of interest. When the ATP procedure is used, excess ATPis removed from the preparation by the addition of apyranase asdiscussed in Levy et al. (1991) Cell 67:265-274, the disclosure of whichis incorporated herein by reference. When the low pH procedure is usedthe buffer is readjusted to neutral pH by the addition of pH modifyingreagents.

The peptide (1mg) and the pretreated stress protein (9mg) are admixed togive an approximate molar ratio of 5-peptides:1 stress protein. Then,the mixture is incubated for 3 hours at room temperature in a bindingbuffer containing 20 mM sodium phosphate, pH 7.2, 350 mM NaCl, 3 mMMgCl1₂, 1 mM PMSF. The preparations are centrifuged through Centricon 10assembly (Millipore) to remove any unbound peptide. The association ofthe peptides with the stress proteins can be assayed by SDS-PAGE andradioautography when radiolabelled peptides are used to reconstitute thecomplexes.

Following reconstitution, the candidate immunogenic stressprotein-peptide complexes can be tested in vitro using for example themixed lymphocyte target cell assay (MLTC) described below. Oncepotential immunogenic constructs have been isolated they can becharacterized further in animal models using the preferredadministration protocols and excipients discussed below.

VII. Determination of Immunogenicity of Stress Protein-Peptide Complexes

The purified and reconstituted stress protein-peptide complexes can beassayed for immunogenicity using the mixed lymphocyte target-cultureassay (MLTC) well known in the art.

Briefly, mice are injected subcutaneously with the candidate stressprotein-peptide complexes. Other mice are injected with either otherstress protein-peptide complexes or whole infected cells which act aspositive controls for the assay. The mice are injected twice, 7-10 daysapart. Ten days after the last immunization, the spleens are removed andlymphocytes released from the excised spleens. The released lymphocytesmay be restimulated in vitro by the subsequent addition of dead cellswhich prior to death had expressed the complex of interest.

For example, 8×10⁶ immune spleen cells may be stimulated with either4×10⁴ mitomycin C treated or γ-irradiated (5-10,000 rads) cells (thecells having been infected with the intracellular pathogen ortransfected with an appropriate gene) in 3 ml RPMI medium containing 10%fetal calf serum. In certain cases 33% secondary mixed lymphocyteculture supernatant may be included in the culture medium as a source ofT cell growth factors. See for example, Glasebrook et al. (1980) J. Exp.Med. 151;876. In order to test the primary cytotoxic T cell responseafter immunization, spleen cells may be cultured without stimulation. Insome experiments spleen cells of the immunized mice also may berestimulated with antigenically distinct cells, to determine thespecificity of the cytotoxic T cell response.

Six days later the cultures are tested for cytotoxicity in a 4 hour⁵¹Cr-release assay. See for example, Palladino et al. (1987) Cancer Res.47:5074-5079 and Blachere et al. (1993) J. Immunotherapy 14:352-356, thedisclosures of which are incorporated herein by reference. In thisassay, the mixed lymphocyte culture is added to a target cell suspensionto give different effector: target (E:T) ratios (usually 1:1 to 40:1).The target cells are prelabelled by incubating 1×10⁶ target cells inculture medium containing 200 mCi⁵¹Cr/ml for one hour at 37° C. Thecells are washed three times following labeling. Each assay point (E:Tratio) is performed in triplicate and the appropriate controlsincorporated to measure spontaneous ⁵¹Cr release (no lymphocytes addedto assay) and 100% release (cells lysed with detergent). Afterincubating the cell mixtures for 4 hours, the cells are pelleted bycentrifugation at 200 g for 5 minutes. The amount of ⁵¹Cr released intothe supernatant is measured by a gamma counter. The percent-cytotoxicityis measured as cpm in the test sample minus spontaneously released cpmdivided by the total detergent released cpm minus spontaneously releasedcpm.

In order to block the MHC class I cascade a concentrated hybridomasupernatant derived from K-44 hybridoma cells (an anti-MHC class Ihybridoma) is added to the test samples to a final concentration of12.5%.

VIII. Formulation and Vaccination Regimes

Once candidate stress protein-peptide complexes have been identifiedthey may be administered either to an animal model or to the intendedrecipient to stimulate cytotoxic T cell responses against thepreselected intracellular pathogen. The stress protein-peptide complexesof the invention may be either stored or prepared for administration bymixing with physiologically acceptable carriers, excipients, orstabilizers. These materials should be non-toxic to the intendedrecipient at dosages and concentrations employed.

If the complex is water soluble then it may be formulated in anappropriate buffer, for example phosphate buffered saline (5 mM sodiumphosphate, 150 mM NaCl, pH7.1) or other physiologically compatiblesolutions. Alternatively, if the resulting complex has poor solubilityin aqueous solvents then it may be formulated with a non-ionicsurfactant such as Tween, or polyethylene glycol.

Useful solutions for oral or parenteral administration may be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990. Formulations may include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration, in particular, may include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, induding, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the stress protein-peptide complexes in vivo.

Formulations for inhalation administration may contain as excipients,for example, lactose. Aqueous solutions may contain, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Oilysolutions may be useful administration in the form of nasal drops. Gelsmay be applied topically intranasally.

The compounds provided herein can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable non toxicexcipients and carriers. In addition the formulations may optionallycontain one or more adjuvants. Preferred adjuvants include, but are notlimited to, pluronic tri-block copolymers, muramyl dipeptide and itsderivatives, detoxified endotoxin, saponin and its derivatives such asQS-21 and liposomes. The present invention further envisages sustainedrelease formulations in which the complex is released over an extendedperiod of time.

The dosage and means of administration of the family of stressprotein-peptide vaccines prepared in accordance with the invention willnecessarily depend upon the nature of the complex, the intracellularpathogen and the nature of the disease in question. The complex shouldbe administered in an amount sufficient to initiate a cytotoxic T cellresponse against the intracellular pathogen. The preferred dosage ofdrug to be administered also is likely to depend on such variables asthe type of disease, the age, sex and weight of the intended recipient,the overall health status of the particular patient, the relativebiological efficacy of the compound selected, the formulation of thecompound, the presence and types of excipients in the formulation, andthe route of administration.

In general terms, the compounds of this invention may be provided in anaqueous physiological buffer solution containing about 0.001 to 10% w/vcompound for parenteral administration. Typical doses range from about0.1 to about 1000 micrograms of complex/kg body weight ofrecipient/immunization; and preferably range from about 0.5 to about 100micrograms of complex/kg body weight of recipient/immunization. It iscontemplated that between about 10 to about 250 micrograms of complexwill be administered per dose to a human subject weighing about 75 kg.These quantities, however, may vary according to theadjuvant-co-administered with the complex.

The vaccines may be administered using standard protocols which include,but are not limited to, intramuscular, subcutaneous, intradermal,intraperitoneal, intravenous, intravaginal, intrarectal, oral,sublingual, transcutaneous, and intranasal administration. Preferablythe recipient should be vaccinated four times at weekly intervals. Ifnecessary, the responses may be boosted at a later date by subsequentadministration of the vaccine. It is contemplated that the optimaldosage and vaccination schedule may be determined empirically for eachstress protein-peptide vaccine using techniques well known in the art.

Various cytokines, antibiotics, and other bioactive agents also may beadministered with the stress protein complexes. For example, variousknown cytokines, i.e., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL11, IL-12, IFNα, IFNβ, IFNγ, TNFα, TNFβ, G-CSF,GM-CSF, and TGF-α may be co-administered with the complexes in order tomaximize the physiological response. It is anticipated, however, thatother but as yet undiscovered cytokines may be effective in theinvention. In addition, conventional antibiotics may be co-administeredwith the stress protein-peptide complex. The choice of suitableantibiotics will also depend upon the disease in question.

EXAMPLE 1 Immunogenicity of Stress Protein-peptide Complexes Isolatedfrom Cells Transfected with a Gene Encoding an Antigenic Determinant

FIG. 1 shows the antigen specific cytotoxic T cell activity ofsplenocytes derived from mice immunized with a gp96-peptide complexharvested from BALB/c fibroblasts transfected with the nucleoprotein(NP) gene from the PR8 influenza virus.

Briefly, gp96-peptide preparations were isolated from BALB/c cellstransfected with and expressing the NP gene of the PR8 influenza virus.The gp96-peptide complex was isolated from 100,000 g supernatant by theunmodifed gp60-peptide complex purification protocol. Then, thepreparations were used to immunize naive BALB/c mice. The mice wereinjected twice subcutaneously with the gp96-peptide complexes at ten dayintervals. The mice were sacrificed and the spleen cells obtained. Thespleen cells were stimulated twice in vitro by the additional lethallyirradiated BALB/c cells expressing the NP gene using the mixed targetlymphocyte culture (MLTC) assay described above. Six days later thecultures were tested for cytotoxicity using the ⁵¹Cr release assay. Inorder to block the MHC type I cascade the spleen cells were incubatedwith the supernatant derived from K-44 hybridoma (containing anti-MHCtype I immunoglobulins) culture.

The cytotoxic activity was assayed by the release of ⁵¹Cr from BALB/cfibroblasts expressing the NP gene (filled circles), BALB/c cellsexpressing the NP gene but treated with the anti-MHC type I antisera(empty circles) and from the syngeneic non-NP transfected cell line 5117(asterisks). The spleens of the mice mmunized with the gp96 complexshowed strong MHC class I-restricted cytotoxic cell activity againstBALB/c cells expressing the NP gene, but not against the syngeneicnon-NP transfected cell line5117. Furthermore, the anti MHC type Iantisera blocked the response. Therefore, it is apparent thatimmunization with a stress protein-peptide complex elicits a specificcytotoxic T cell response against the peptide in the complex and thatthe MHC class I cascade plays an integral role in stimulating thecytotoxic T cell response against cells infected with intracellularpathogens.

EXAMPLE 2 Immunogenicity of Stress Protein-peptide Complexes Isolatedfrom SV40 Transformed Cells

FIG. 2 shows the antigen specific cytotoxic T cell activity ofsplenocytes derived from mice immunized with gp96-peptide complexharvested from SV40 transformed SVB6 cells.

Briefly, gp96-peptide complexes were isolated from SV40 transformed SVB6cells and used to immunize naive (57BL/6) mice. The gp96-peptide complexwas isolated from 100,000 g supernatant by the unmodifed gp60-peptidecomplex purification protocol. The mice were injected twicesubcutaneously with the complex at ten day intervals. The mice weresacrificed, the spleen cells isolated and stimulated in vitro by theaddition of lethally irradiated SV40 transformed SVB6 cells by the MLTCprocedure. Six days later the cells were assayed for cytotoxicity usingthe ⁵¹Cr release assay. The cytotoxic activity was assayed by therelease of ⁵¹Cr from SVB6 cells (filled triangles) and from a non SV40transfected syngeneic cell line, MCA (empty triangles). MHC class Imediated activity was assayed also by adding anti-MHC class Iinimunoglobulins derived from the K-44 hybridoma cell line to the spleencells.

The spleen cells isolated from mice immunized with the gp96-peptidecomplex showed strong MHC dass I-restricted activity against the SV40transfected SVB6 cells but not against the non transfected cells.

EXAMPLE 3 Reconstitution of Immunogenic Stress Protein-peptide ComplexesIn Vitro

FIGS. 3A-3D show antigen specific cytotoxic T cell activities ofsplenocytes derived from two mice immunized with reconstitutedHsp70-peptide complex.

Briefly, uncomplexed Hsp70 was purified by the procedure described aboveand the peptide (SLSDLRGYVYQGL, SEQ. ID. NO.:1) was synthesized by solidphase peptide synthesis. The peptide (1 mg) and ATP treated Hsp70 (9mg)were admixed and incubated for 3 hours at room temperature in a bindingbuffer containing 20 mM sodium phosphate, pH 7.2, 350 mM NaCl, 3 mMMgCl₂, 1 mM PMSF. The resulting preparation was centrifuged throughCentricon 10 assembly (Millipore) to remove unbound peptide.

The resulting complex was used to immunize two naive mice. The spleencells were isolated from the mice and stimulated twice in vitro by theaddition of lethally irradiated EL4 cells transfected with, andexpressing a minigene encoding the peptide SLSDLRGYVYQGL (SEQ. ID.NO.:1), using the MLTC procedure. The cytotoxicities of spleen cellsfrom both mice were assayed after the first (3A and 3C) and second (3Band 3D) stimulations by the ⁵Cr release assay. The release of ⁵Cr wasmeasured from EL4 cells (hollow triangles) and from EL4 cellstransfected with, and expressing the peptide SLSDLRGYVYQGL (SEQ. ID.NO.:1) (filled triangles). The results show that stress proteins andpeptides can be reconstituted successfully in vitro to give specificimmunogenic stress protein-peptide complexes.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

1 13 amino acids amino acid single linear peptide Peptide 1..13 /label=PEPTIDE1 /note= “ANTIGENIC PEPTIDE I” 1 Ser Leu Ser Asp Leu Arg Gly TyrVal Tyr Gln Gly Leu 1 5 10

What is claimed is:
 1. A composition comprising an amount of a purifiedpopulation of peptides, wherein said purified population of peptides isproduced by a method comprising the steps of: (a) purifying a populationof non-covalently associated stress protein peptide complexes from amammalian cell infected with an intracellular pathogen; (b) eluting thepeptides from said population of complexes to produce dissociatedpeptides; and (c) recovering the dissociated peptides.
 2. Thecomposition of claim 1, further comprising a cytokine.
 3. Thecomposition of claim 2 wherein said cytokine is selected from the groupconsisting of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IFNα, IFNβ, IFNγ, TNF∝, TNFβ, G-CSF, GM-CSF,GM-CSF and TGF-β.
 4. The composition of claim 1, wherein said mammaliancell is an immortalized mammalian cell.
 5. The composition of claim 1,wherein said stress protein is a human stress protein.
 6. Thecomposition of claim 1, wherein said pathogen is a bacteria.
 7. Thecomposition of claim 6, wherein said bacteria is selected from the groupconsisting of Mycobacteria, Rickettsia, Mycoplasma, Neisseria, andLegionella.
 8. The composition of claim 1, wherein said pathogen is aprotozoan.
 9. The composition of claim 8 wherein said protozoan isselected from the group consisting of Leishmania, Trypanosoma andKokzidioa.
 10. The composition of claim 1, wherein said pathogen is anintracellular parasite.
 11. The composition of claim 10 wherein saidparasite is selected from the group consisting of Chlamydia andRickettsia.
 12. The composition of claim 1, wherein said pathogen is avirus.
 13. The composition of claim 12 wherein said virus is selectedfrom the group consisting of influenza, hepatitis type A, hepatitis typeB, hepatitis type C, varicella, adenovirus, herpes simplex type I(HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus,echovirus, rotavirus, respiratory syncytial virus, papilloma virus,papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus,Coxsackie virus, mumps virus, measles virus, rubella virus, polio virus,human immunodeficiency virus type I (HWV-I), and human immunodeficiencyvirus type II (HIV-II).
 14. The composition of claim 1, wherein thestress protein is a member of the stress protein families selected fromthe group consisting of hsp60, hsp70, hsp90 and a combination thereof.15. The composition of claim 14, wherein the stress protein is gp96. 16.The composition of claim 15, wherein said stress protein is heat shockprotein
 70. 17. The composition of claim 1, which further comprises apharmaceutically acceptable carrier.
 18. The composition of claim 1,further comprising an adjuvant.
 19. The composition of claim 13 whereinthe adjuvant is selected from the group consisting of pluronic tri-blockcopolymers, muramyl dipeptide, detoxified endotoxin, saponin, QS-21, andliposomes.
 20. A composition comprising an amount of a purifiedpopulation of peptides, wherein said population of peptides ischaracterized as being present as non-covalent complexes with aplurality of heat shock protein 70 polypeptides in mammalian cellsinfected with an intracellular pathogen.